ABSTRACT
Viral infectious diseases may cause neurological symptoms primarily in two nonexclusive ways. Infection may lead to an excessive inflammatory response that damages the neuronal system, which is referred to as immunopathology, or the pathogen is able to infect brain cells, such as neurons or nonneuronal glial cells, like oligodendrocytes, microglia, and astrocytes. Viruses that target and infect cells of the CNS, in general, are called neurotropic. 3D brain organoids provide favorable conditions to study target cells and induced pathomechanisms associated with such viral neurotropism. In the context of highly pathogenic viruses, strict safety precautions (safety level-3 laboratory) must be taken if infectious laboratory strains or strains from clinical samples are to be used for infection experiments. Likewise, safe inactivation protocols must be used for subsequent analysis. This chapter will discuss appropriate protocols, focusing on methodological aspects for each of these steps, and discuss advantages and disadvantages when working with 3D brain organoids while handling biosafety level-3 pathogens based on our work with severe acute respiratory virus type 2 (SARS-CoV-2). Copyright © 2023, Springer Science+Business Media, LLC, part of Springer Nature.
ABSTRACT
Covid-19 testing is not accessible for millions during this pandemic despite our best efforts. Without greatly expanded testing of asymptomatic individuals, contact tracing and subsequent isolation of spreaders remains unobtainable as a means for control. In an effort to increase RT-PCR assay testing for the presence of the novel beta-coronavirus SARS-CoV-2 as well as improve sample collection safety, GenTegra LLC has introduced two products for saliva collection and viral RNA stabilization: GTR-STM (GenTegra Saliva Transport Medium) and GTR-STMdk (GenTegra Saliva Transport Medium Direct to PCR). Both products contain a proprietary formulation based on GenTegra's novel "Active Chemical ProtectionTM" (ACP) technology that gives non-dilutive, error-free saliva sample collection using RNA stabilization chemicals already dried in the collection tube. GTR-STM can be used for safer saliva-based sample collection at home (or at a test site). Following saliva collection, the sample-containing GTR-STM can be kept at ambient temperature during shipment to an authorized CLIA lab for analysis. SARS-CoV-2 viral RNA in GTR-STM is stable for over a month at ambient temperature, easily surviving the longest transit times from home to lab. GTR-STM enhances patient comfort, convenience, compliance and reduces infectious virus exposure to essential medical and lab professionals. Alternatively, the GTR-STMdk direct-into-PCR product can be used to improve lab throughput and reduce reagent costs for saliva sample collection and testing at any lab site with access to refrigeration. GTR-STMdk reduces lab process time by 25% and reagent costs by 30% compared to other approaches. Since GTR-STMdk retains SARS-CoV-2 viral RNA stability for three days at ambient temperature, it is optimized for lab test site rather than at home saliva collection. SARS-COV-2 viral RNA levels as low as 0.4 genome equivalents/uL are detected in saliva samples using GTR-STMdk. This increased sensitivity of SARS-CoV-2 detection can expand Covid-19 testing to include asymptomatic individuals using pooled saliva. The formal Limit of Detection (LoD) of SARS-CoV-2 in GTR-STM and GTR-STMdk in human saliva in individual and pooled samples will shortly be known.